Method for preparing tissue, particularly adipose tissue, for transplantation from lobular fat extracted by liposuction

ABSTRACT

A method of preparing adipose tissue for transplantation, from lobular fat extracted, for instance, by liposuction, said fat consisting of a fluid component comprising an oily component, a blood component and/or sterile solutions and of a solid component comprising cell fragments, cells and one or more cell macroagglomerates of heterogeneous size, characterized in that it includes at least one step of washing the cell aggregates carried out at the same time as a step of separating the fluid component, in emulsion form, from the solid component.

This application is a continuation of U.S. application Ser. No.14/700,186, filed on Apr. 30, 2015, which is a continuation of U.S.application Ser. No. 13/698,400, filed Jan. 22, 2013, now U.S. Pat. No.9,044,547, which is a 35 U.S.C. § 371 U.S. National Stage application ofInternational PCT Application No. PCT/IB2011/052204, filed May 20, 2011,which claims priority to Italian Patent Application No. GE2010A000057,filed May 20, 2010.

The present invention relates to a device and a kit for preparingtissue, particularly adipose tissue, for transplantation, from lobularfat extracted by liposuction.

The invention also relates to a method of treating body and face volumedeficiencies, improving skin trophism and/or for biological stimulationby the adipose tissue obtained through said method.

According to the prior art, the preparation required to reuse theliposuctioned material involves the separation of the vital cellcomponent to be reinjected from the waste material composed ofanesthetic liquid or biological fluids (serum or blood) and cell debrisand oil resulting from the rupture of suctioned adipocytes.

Such separation may occur within the syringe that is used forwithdrawal, or in special containers, essentially in three manners:

-   -   by settling: the materials separate by differences in density        under gravity,    -   by centrifugation: the materials separate by differences in        density under the effect of centrifugal force,    -   by washing: the lipoaspirate is placed in a thin-mesh strainer        and washed, generally with a saline, that may be progressively        replaced or not.

According to the best known technique (Coleman lipostructure), thesyringes containing the lipoaspirate are closed at the bottom by aluer-lock cap, and are placed in a centrifuge for separating the liquidphase from the solid biological material.

Before using the biological material so obtained, the anesthetic andbiological liquids left on the bottom of the syringe aftercentrifugation shall be manually drained, by removing the luer-lock capfrom the syringe and causing them to flow out by gravity, whereas thecell fragments and oil resulting from the break of the cell wallsof-adipocytes lie on the cell material to be transplanted and areremoved in an incomplete and rudimentary manner, using gauzes thatpartially absorb the excess oil and often make the last part of thesuctioned material unusable.

The above described technique suffers from certain drawbacks.

First, the step of suction and separation by centrifugation causes aconsiderable amount of adipocytes to break and release much oil, whichcannot completely removed with the Coleman technique, and makes asignificant portion of the lipoaspirate unusable, i.e. the portion ofcell material that, after centrifugation, is located on the upper partof the syringe barrel, in contact with oil, and hence is contaminated bysaid oil.

This is because the presence of oil in the biological filler to beinjected increases the risk of infections and rejections and causesincreased inflammation.

Furthermore, the above described process involves multiple contacts ofthe liposuctioned material with surfaces of various types ofinstruments, as well as long-time contact with air in a potentiallynon-sterile environment, whereby use thereof in an operating room isrecommended.

A technique is also known but rarely used, which involves mechanicalfragmentation of the suctioned cell agglomerate using a blender, whosecutting blades separate fat lobules and provide an injectable cellsuspension.

This fragmentation technique has many drawbacks.

First, the fragmentation step, which is followed by centrifugation,causes a considerable amount of adipocytes to break, which causes morethan half of the liposuctioned material to be unusable for lateraesthetic treatments. As a direct result, an increased number ofliposuction sessions are required to compensate for this loss ofmaterial occurring during preparation of the material to betransplanted, with increased discomfort for patients.

Furthermore, the quantity of usable cell suspension that can be obtainedusing the above described procedure and devices largely depends on theskill of the health care staff in setting the speed and operating timeparameters of the blender and the centrifuge and on the conditions ofthe instruments: an excessive rotation speed of the blades or the use,for example, of a blender with poorly cutting blades does not causeseparation of fat lobules, but rather the mechanical break of the cellwalls of a large amount of adipocytes, which involves oil formation andmakes the cell suspension unusable, in addition to requiring accurateseparation of the cell fragments and oil from the suspension. This isbecause the presence of oil in the biological filler to be injectedincreases the risk of infections and rejections.

Furthermore, the above described process involves multiple contacts ofthe liposuctioned material with surfaces of various types ofinstruments, as well as some contact with air in a non perfectly sterileenvironment, as is the case of doctor's offices. Since the material isof biological nature, extended contact with air or with multipleinstruments, that may even not be perfectly sterile, increases the riskof bacterial or viral contamination, and may jeopardize treatmentresults.

The technique that involves washing through a strainer also has certaindrawbacks.

Particularly the strainer net may easily become clogged with theliposuctioned material, which requires a. manual action to remove fatfrom. The meshes, thereby slowing down the preparation process andespecially increasing the risk of contamination of the material to beinjected.

The use of a simple strainer does not allow the liposuctioned materialto be constantly maintained in a closed and perfectly sterileenvironment throughout the preparation process, i.e. from theliposuction step to the injection step.

Patent documents are known which disclose cell isolating devices.

The international application WO 2009/073724 discloses a method and anapparatus for isolating cells from lipoaspirate.

Particularly, it discloses a method for separating the adipocyte and oilfraction from the non-fat cell fraction in a lipoaspirate.

In order to obtain lipids and adipocytes that float on a cell solutionof interest and other small cells within a container defined as“separation chamber”, the adipose tissue is placed in a digestionchamber, and forced through a filter and through a head having poresinto said “chamber”.

The steps of washing the tissue, removing excess liquids, enzymaticdigestion, antibiotic addition and cell selection may occur in acontainer defined as “digestion chamber”. The digestion chamber maycontain a filter that retains the tissue but allows the passage ofdissociated cells and fluids. An aqueous emulsion containing adipocytelipids is formed in this chamber.

The dissociated material in the digestion chamber may pass through adispersing filter with pores smaller than the pores on the dispersinghead contained in the first “separation chamber”. This filter 115 isused to prevent clogging of the pores of the dispersing head.

In the “separation chamber” the lipids and adipocytes are separated fromthe cell population.

The device provides a cell population from a tissue without using thecentrifuge but by forcing the solution through filters with pores ofvarious sizes.

Said device is particularly complex in terms of construction, as shownin the figures.

Furthermore, the many passages of the organic material through chambersand filters extend the duration of the method, and expose the organicmaterial to contamination risks.

Also, the complexity of the method and device make them unsuitable foruse, for instance, in out-patient environments, which require quickpreparation of injectable material from lipoaspirate and quickperformance of face and body defect correction without the assistance ofparticularly specialized staff.

Furthermore, in this method, emulsions are formed using chemicals andnot only through the use of mechanical means and forces.

US Application 2007/0274960 discloses a method of preparing a stemcell-containing composition. In order to prepare a stem cell population,in certain embodiments the liposuctioned adipose tissue is physicallytreated, i.e. cut or minced into smaller pieces, and undergoes enzymatictreatment, which facilitates release of the cells of interest from theother tissue components.

Therefore US 2007/0274960 allows the adipose tissue to be divided intosmaller pieces by forcing it through an array of screens, to obtainsmaller portions of uniform sizes, that can undergo enzymatic treatmentin a more uniform manner, thereby providing a quicker release of stemcells and reducing the contact time between the released cells and theenzyme solution.

According to this patent, an emulsion of adipose tissue may be preparedusing a perfluorocarbon solution, which emulsion is separable from thestem cells of interest.

The patent does not include the preparation of an emulsion of liquidsthat can be mechanically separated from lipid cells or small cellagglomerates. The container that contains the cutting means cannot bealso used for injecting adipose tissue into a patient. U.S. Pat. No.6,020,196 discloses a method for collecting microvascular endothelialcells.

The patent describes a method of treating suctioned adipose tissue,which adipose tissue, suctioned by a syringe with a cannula havingapertures of such a size as to minimize stresses on cell components andto obtain a homogeneous adipose tissue, is forced from one syringe toanother through a filter ('74) located between the suction ports of thetwo syringes.

By pulling the pistons of the syringes, the suctioned adipose tissue ishomogenized by being forced through the filter from one syringe toanother.

A lower viscosity of the suctioned material allows easier removal of thecontaminants and improved digestion of the sample, for obtainingendothelial cells.

The method as disclosed in this patent suffers from certain drawbacksthat make it unsuitable for use in the preparation of injectable fat,because:

-   -   the filter may become clogged by the adipose tissue: the        filter-holding device forms a restriction in the flow line from        one syringe to the other; the clogged filter obstructs the        passage of adipose tissue from one syringe to the other and        requires disconnection of the syringe and replacement of the        filter to continue adipose tissue washing; due to these steps,        the preparation of an emulsion of solid and liquid components        becomes difficult and time-consuming and the organic material is        exposed to contamination;    -   the passage through the filter meshes for disintegration of the        connective tissue also leads to the break of adipocytes, with        formation of excess oil and the need for a later accurate        separation of intact fat cells from oil.

U.S. Pat. No. 6,020,196 provides a homogenate from which endothelialcells may be extracted with the addition of collagenase andcentrifugation, hence through the combination of chemical and physicalactions. The patent does not involve the formation of an emulsion ofliquid components upon which lipid cells or small agglomerates of lipidcells obtained from liposuctioned adipose tissue may float, which cellsare directly injectable, after appropriate treatment, into a patient,without requiring particular sterile conditions of the environments,e.g. without requiring a perfectly sterile operating room.

U.S. Pat. No. 6,020,196 does not involve the possibility of providing asingle device that, through a few simple treatment steps, allowspreparation of the liposuctioned material and collection and temporarystorage of fat, until reinjection.

Patent application US 2003/0100105 discloses an apparatus for extractingcells from organs. The apparatus includes a digestion chamber containingthe organ and protease, and agitation means, such as balls having atleast one cavity, which balls only act upon the organ.

Therefore, like the previous patents, this patent involves a chemicallyaggressive treatment of the organic material.

As described in column 3, paragraph 0026, the agitation means 14 aremoved with the digestion chamber 12 to agitate the organ and facilitatethe release of the cells.

The patent does not involve the possibility of obtaining an adiposetissue preparation for transplantation from lobular fat, particularlythe patent involves the use of balls to agitate the organ and facilitaterelease of cells from said organ.

The object of the present invention is to provide a simple andinexpensive device, capable of obviating the above drawbacks, forpreparing tissue, particularly adipose tissue for transplantation fromlobular fat, i.e. fat composed of cell macroagglomerates, cells and cellfragments, which fat is obtained by means of liposuction.

The object of the present invention is to provide a device that canprovide tissue for transplantation without “using” chemicals forpreparation, i.e. with no chemical aggression or any other chemicaltreatment of the lipoaspirate. This provides a simple and easy-to-usedevice, which only uses manually applied mechanical forces, which devicemay not require trials for medical use, or only require simple trials.The device of the present invention may also be used in medicalout-patient environments, without requiring particular sterileconditions, such as the conditions required in an operating room.

A further object of the present invention is a method that involves theuse of said device for preparing tissue, particularly adipose tissue fortransplantation, said method and device allowing the biological materialto be maintained in a wholly closed system, i.e. a system that preventsany contact of the suctioned patient material with the outsideenvironment.

Another object is also a method of treating body and face volumedeficiencies, improving skin trophism and/or for biological stimulationby the adipose tissue obtained through said device and method.

Particularly, the device of the present invention allows preparation ofcell agglomerates, particularly adipocyte agglomerates, using a fewsimple instruments and a few processing steps, without using chemicalsor physico-chemical treatments, but only mechanical stirring, whileeliminating most of the oily component and avoiding handling of thebiological material in a non-perfectly sterile environment.

Thus, also due to the use of specially thin needles, the transplantationof adipose tissue will be less invasive, less traumatic and moreeffective.

Furthermore, the cell agglomerates yielded by the device of the presentinvention are prepared with minimized or no contact with the outsideenvironment and using disposable instruments that reduce the risks ofcontamination of the biological material, the risks of instrumentdeterioration and the drawbacks associated with washing andre-sterilization.

The biological material so obtained may be injected into any tissue ororgan.

The above objects are fulfilled by a device composed of at least onewashing and separating container having a washing chamber for washingthe liposuctioned material, which container has an inlet and an outletfor the liposuctioned material to enter the washing chamber through theinlet and for at least part of said material, particularly the fluidcomponent, to exit said chamber through the outlet, said washing chamberincluding means for mechanically forming an emulsion of fluidcomponents, on which the cell components designed to be used for latertransplantion will float, separate from the liquid component.

Preferably, the tissue so prepared is used for autotransplantation,although the device may be also used for preparing tissues forallotransplantation.

The device and method of the present invention can provide not onlyadipose tissue for use as a biological filler, i.e. for correction offace and body volume deficiencies, but also macroagglomerates of adiposetissue having stem cells on their surface, whose arrangement in contactwith the tissue of the injection area, allows quick regeneration of thetreated tissues.

These means for mechanically forming an emulsion are capable ofobtaining an emulsion of blood fluids, blood residues, oils and othersolutions (such as washing saline solutions or anesthetic solutions usedduring suction), contained in the liposuctioned material, by simplemechanical action, allowing said fluids to remain separate from thesolid cell material, i.e. lipid cells, stem cells.

The separation of the liquid phase to be eliminated from the solid phaseto be transplanted is only obtained by a mechanical (and not a chemical)action.

In the preferred embodiment, this action is performed by stirring means,that may be of any type, particularly of active or passive type.

Active means are motorized stirring means, driven by a motor or a motiveforce to provide the stirring movement.

The passive means are means that exert their action upon stirring of thecontainer, and hence operate, for example, by inertia.

The stirring means form an emulsion of the liquids to be eliminated andparticularly fatty liquids, in a solvent such as a physiologic washingfluid. Especially in the embodiment with passive stirring means, thedevice has a very simple construction and is effective.

With this device, the mechanical action of the container, in combinationwith passive stirring means will not be of such strength as to requirethe use of mechanical means. Manual stirring is sufficient in thepresent device.

Preferably, the mechanical stirring action consists in a rotation of thecontainer about an axis, e.g. a longitudinal axis, perpendicular to theend surfaces of the container, and either external or internal to thecontainer. Other kinds of stirring may be also provided, such as shakingor the like.

These means for forming an emulsion by simple manual or possiblymechanical stirring of the washing and separating container, withoutusing chemicals or enzymes that might lead to disintegration of the cellmaterial, afford separation of the solid component from the liquidcomponent in the washing chamber, and particularly allow the solidcomponent, consisting of cell fragments, cells and cell aggregates, tofloat on an emulsion of liquid substances, such as blood, sterilesolutions and oil yielded from broken fat cells. In a preferredembodiment, the solid component is washed in the washing chamber: asterile washing solution, e.g. a sterile saline, is injected once ormultiple times into the washing and separating container through theinlet. With stirring of the container, this solution allows the cellmaterial for transplantation to be cleaned of any waste liquid, such asblood and oil.

The suctioned fat is composed of a mixture of fluid materials and cellfragments, cells and one or more cell macroagglomerates of heterogeneoussize.

The emulsion formed upon stirring, due to the presence of mechanicalemulsifying means in the washing chamber, is caused to exit from theoutlet and be collected in a sealed container, to prevent contaminationof the outside environment as well as to obtain cell material (cellfragments, cells, cell agglomerates) for transplantation, stored in thewashing chamber in perfectly sterile conditions.

Then, said cell material is caused to exit the washing and separatingchamber, and be injected or divided and stored, for latertransplantation, into one or more sterile containers, such as syringesor the like.

Therefore an emulsion of waste liquids is simply formed, on which cellfragments, lipid cells, lipid microaggregates and stem cells float,which fragments or cells will be ready for use with no additionaltreatment, particularly with no chemical treatment.

The washing and separating container will be filled with lipoaspirate upto about ⅓ of its volume, the rest of the volume being filled withwashing liquid.

There is no air in the container during treatment of the material.

The fat and/or the fat washing or treating liquids are forced throughthe device of the present invention by applying pressure or suction onthe contents of said washing and separating and/or size reducingcontainers, i.e. on the material to be treated, through the use ofcompression means such as syringes connected to said containers, pistonsthat cooperate with the openings of said containers or the like.

Therefore, the device allows washing of the lipoaspirate and separationof the cell mass from the emulsion of fluid materials, such as washingsolution, saline solution, anesthetic solution, blood and oil, so that aminimized amount of undesired impurities are collected at the end of theprocedure with the cells or cell aggregates, particularly adipocytes.

With the device of the present invention, an emulsion is formed bymechanical means and particularly by washing, with elimination of theemulsion of liquid components through a density gradient.

The device may be used for washing lipoaspirate divided into smallercells and/or agglomerates.

Techniques are known for dividing the liposuctioned material, e.g. byusing a blender.

In a preferred embodiment, the liposuctioned material, particularly cellmacroagglomerates are reduced to smaller sizes for easiertransplantation.

According to the invention, in the washing and separating container orin another container, known as size reducing container, which is adaptedto be fluid-tightly connected to said washing and separating container,size reducing means are provided for reducing the size of the solidcomponent of the lipoaspirate, particularly cell magroagglomerates, toaveragely equal smaller cell agglomerates, having a size equal to orsmaller than a given value, which means consist of at least one seriesof parallel or intersecting sheets or cutting wires, to form at leastone size reducing net, through which the liposuctioned material ispassed.

In a preferred embodiment, homologation and/or size reduction of thelipoaspirate occurs before washing, by means of a first size reductionnet, through which the liposuctioned material is forced before enteringthe washing chamber of the washing and separating container, and asecond size reduction/homologation occurs by means of a second sizereducing net, which size reduction is performed after at least one cellmaterial washing step in the washing and separating container.

The second size reduction/homologation may occur at the end of thewashing steps, before the exit of the material for transplantation fromthe washing and separating container.

Preferably the second size reducing net has narrower meshes than thefirst size reducing net.

Obviously, the material for transplantation may be caused to passthrough multiple nets or size reducing means, particularly through twoor more nets or size reducing means, said size reducing means beingpossibly provided in a single container or in two or more connectablewashing and separating containers.

The first reduction facilitates washing as it fragments or stops thefibrous component of fat lobules and it homologates the size of thelipoaspirate mass by reducing it into smaller agglomerates, separatefrom each other and from the lipoaspirate itself. The second reductionprovides a washed cell material ready for transplantation, whose sizeallows injection by any kind of needle, even of very small size.

The presence of at least two size reducing means, having meshes orapertures of different sizes, particularly larger meshes or apertures inthe net located at the lipoaspirate inlet and smaller meshes in the netlocated at the outlet for the material ready for transplantation, andthe provision of a given distance between said two means, i.e. at theend sides of the washing chamber, prevent said size reducing means frombeing clogged with the cell material, as size reduction occursgradually.

Furthermore, such gradual size reduction of the lipoaspirate allowslarge needles to be possibly used during withdrawal of the tissue fromdonor areas, thereby speeding up the material collection procedure.Therefore, even when the liposuctioned material is composed of largeagglomerates, it still doesn't clog the apertures or size reducing meansof the device, as it is reduced into progressively smaller agglomerates,not by passing through a single size reducing mesh, net or sheet, but bysequentially passing through two or more size reducing means with meshesor apertures of decreasing sizes in the direction of the flow of thematerial to be treated.

Preferably, the cell material is reduced to a size allowingtransplantation thereof through very thin needles after the end of thewashing step/s.

The use of transplantation cannulae of particularly small size reducesthe trauma caused by the transplantation procedure, and allows thelatter to be performed under local anesthesia, with no suture orparticular medication and with fast healing results.

Considering that the liposuctioned material also contains stem cells,fat size reduction provides small cell agglomerates, particularlymicroagglomerates of fat cells or individual cells, having stems cellsadhered on their surface. The reduction of the lipoaspirate mass intomany agglomerates provides a larger amount of stem cells potentiallycontacting the tissue to be treated upon injection of the materialprepared by the method and the device of the present invention, as thesize reducing action, in addition to reducing the suctioned mass intomany equal smaller agglomerates, also causes an increase of the surfacearea potentially contacting the tissue to be treated, thereby increasingthe exposure area with stem cells thereon.

The transformation of lobules of adipose tissue yielded by liposuctioninto a biological filler, i.e. a cell suspension or mass or a fluid orsemifluid agglomerate containing adipocytes, other types of cells, suchas stem or mesenchymal cells, and possibly cell fragments and residuesof connective material, which suspension, at the end of thetransformation procedure of the present application, has a solid phasecomposed of cells and/or cell aggregates of small, averagely homogeneoussizes, adapted to be injected in small or large amounts, allows theprepared fat to be used not only in an intra- or submuscular injection,but also in subcutaneous injections, without irregularities, hardeningeffects, calcifications and total reabsorption of the injected fat.

Nevertheless, the fat material may be injected into any tissue or organ.

Furthermore, the separation of fat lobules into small cells or cellagglomerates facilitates engraftment, i.e. integration of the cell massin the tissues in which it is injected.

The division of fat lobules into cell aggregates also provides anincrease of the surface of the injected cell mass that contacts thetissues undergoing transplantation, thereby promoting biologicalstimulation of treated tissues and hence integration of the injectedcell material.

The use of a simple device, composed of a few sterile components, whichisolate the extracted biological material from the external environmentactually throughout the preparation procedure, considerably reduces therisk of contamination of the biological material, the personnel and theenvironment, and. hence the risk of infections or rejections duringlater use of the cell material.

Therefore, the device of the present invention also allows treatment ofbiological material outside operating rooms, in outpatient settings.

The simple construction of the device and the lack of any chemicaland/or enzyme treatment of the lipoaspirate, except washing solutionsleading to the formation of emulsions, can facilitate production andsales, and can avoid, facilitate or reduce the long and. expensiveresearch steps conducted to collect safety and effectiveness data aboutnew drugs or new devices to obtain authorizations for use thereof.

Thus, the device and the method of the present invention afford a simpleand quick procedure, for instance requiring a single outpatient session,for suction of fat from the body of a patient, treatment of said fat,with no chemical/enzyme emulsion of the lipoaspirate, and storage and/orreinjection thereof into a patient.

With the device of the present invention, having means therein,particularly balls, for forming an emulsion of liquids, a very shorttime is required to obtain emulsion of the liquids and separation of thesolid cell material floating on said emulsion, by manual mechanicalstirring.

The time required for treating the lipoaspirate and make it suitable foruse is averagely 10 to 20 minutes; in any case, the time required forobtaining transplantable material is shorter than required with otherprior art devices or methods.

The lipoaspirate storage and treatment device may be also used for thelater transplantation step.

Otherwise, preferably, the treated material may be divided andtransferred to other containers, for instance one or more 10 ccsyringes, which containers may undergo settling and/or centrifugation,to separate the residual liquid/oily component from the solid component,which will be thus ready for transplantation, by subsequent transferinto one or more syringes of less capacity.

Therefore, the device of the present invention affords quick sizereduction of adipose tissue clusters, allowing them to be injectedthrough very small needles, leaving no visible scar on the patient (theprepared tissue may be used for face transplantation, where no large andvisible holes would be aesthetically acceptable), and separate theemulsion/cell phases, to obtain injectable cells or cell agglomerateshaving a larger surface for the stem cells contained in thelipoaspirate, in contact with the tissues to be treated Reduction intosmall agglomerates allows activation of the peripheral stem cells lyingon the outer surface of said agglomerates. The more agglomerates, thelarger the exposed cell surface and the more peripheral stem cellspotentially interacting with the treated tissues.

The method, device and kit as described in greater detail below may beused for treating not only lipid material but any kind of cell aggregaterequiring cell aggregate size reduction and/or washing and separation ofthe liquid phase from the solid cell phase, simply by mechanicalstirring.

These and other features and advantages of the present invention willappear more clearly from the following description of a few embodiments,illustrated in the annexed drawings, in which:

FIG. 1 is a perspective view of the washing and separating containeraccording to one embodiment,

FIG. 2 is a perspective view of the washing and separating containeraccording to a different embodiment,

FIG. 3 is a side view of the washing and separating container,

FIG. 4 is a longitudinal sectional view of the washing and separatingcontainer, with a filter located near the outlet,

FIG. 5 is a longitudinally sectional view of the washing and separatingcontainer, having: a cell agglomerate size reducing net located near theinlet of the washing chamber, a second size reducing net located nearthe outlet, and stirring elements in the washing chamber,

FIG. 6 is a perspective view of the size reducing container,

FIG. 7 is a longitudinally sectional view of the size reducing containerhaving a size reducing net near the inlet and a collection chamber forthe reduced-size fat,

FIGS. 8a-8d show the lipoaspirate treatment steps occurring in thewashing and separating container,

FIG. 9 is a longitudinally sectional view of the washing and separatingcontainer with the solid component floating on the emulsion of liquidcomponents to allow the liquid component to exit from the outlet of thewashing chamber,

FIG. 10 shows the device of the present invention in the foreground,connected to ambulatory and/or surgical equipment,

FIGS. 11a, 11b, 11c show embodiments of the size reducing means,

FIGS. 12a, 12b show and embodiment of a size reducing net, with anenlarged detail,

FIGS. 13a, 13b, 13c show and embodiment of the washing and separatingcontainer and the closing terminal,

FIG. 14 shows a needle adapted for use in combination with the device,for transplantation of the prepared material.

The device of the present invention provides a cell suspension ofadipose tissue to be used as a biological filler, i.e. a filler ofnatural and autologous or heterologous origin, during body and facevolume correction procedures and/or during biological stimulation of anytissue or organ injected with said treated adipose material alone orwith any other synthetic or natural filler.

The lobular fat material, i.e. the macroagglomerates of cells,particularly adipocytes, that are treated by the device of the presentinvention, may be obtained by means of a liposuction procedure whichinvolves extraction of adipose tissue from any donor area of thepatient, e.g. subcutaneous hip, abdomen or knee areas, under localanesthesia or generally in outpatient settings.

Once said adipose tissue has been treated with the device of the presentinvention, it can be used for autotransplantation, i.e. injection intospecial areas of the body of the patient from which the tissue has beenwithdrawn, to fill areas that, due to aging, diseases, treatments, e.g.radiotherapy; or past surgery, exhibit volume deficiencies orreabsorption of subcutaneous fat, with the relevant part of the bodybeing sunken, with projecting bones and sagging skin.

The tissue treated with the device and method of the present inventionmaybe arranged to be used on receiving patients other than the donorpatient.

An exemplary liposuction procedure will be now described.

The liposuction procedure includes preparation of the anesthetic liquid,appropriately diluted and possibly added with vasoconstrictiveadrenaline, in sterile syringes, preferably having a needle or cannulaconnector known as luer connector, with the barrel volume suitable forthe surface area of the body to be anesthetized, for instance 10 to 60cc syringes.

Once the areas for withdrawal of adipose tissue have been marked andsuitably disinfected, the local anaesthetic liquid is injected throughsterile disposable blunted-end cannulas of about 1 mm diameter,preferably having a luer connector.

The patient's skin is sterilized with conventional techniques andanesthetized by a wheal.

Anesthesia is obtained by introducing the anesthetic solution throughprogressive introduction of the disposable blunted-end sterile cannulathrough the skin into the subcutaneous tissue to impregnation of theentire target area with the anesthetic.

For anaesthetic injection and later insertion of the needle or cannulafor adipose tissue withdrawal, the skin may be perforated by a sterilepointed needle or a sterile lanceolate blade of sufficient diameter toallow later introduction of the disposable withdrawal cannula.

If needed, the patient may be sedated.

Liposuction, i.e. withdrawal of adipose tissue is performed with asterile syringes connected to a cannula, preferably having multipleholes with a diameter ranging from 1.2 to 3 mm. Cannulas of up to 6 mmdiameter may be also used.

With the device of the present invention, large diameter cannulas(preferably 3 mm multi-hole cannulas) may be used for withdrawal,allowing quick extraction of a large amount of adipose tissue, becausethe macroagglomerates of cells so extracted will be subjected to sizereduction before use, as described in greater detail below. In oneembodiment, said syringe, having at least a 10 cc volume, has a luerconnector for connection to a disposable sterile cannulas, whichcannulas have one or more holes over their surfaces for suction of thetissue.

Obviously, the volume of the suction syringe and the suction cannuladepends on the amount of adipose tissue to be suctioned, which in turndepends on the volume of treated adipose tissue that is deemed to berequired for the later filling and/or correction procedure.

The adipose tissue withdrawing cannula is preferably introduced throughthe holes formed in the skin beforehand for anesthesia.

10 cc syringes are preferably used for liposuction, as they exert anadequate pressure on the tissues and allow easy handling.

These are preferably the known luer lock syringes 7, which are connectedto a suction cannula having a corresponding luer connector, directly orwith the interposition of special two-way valves between the cannula andthe syringe, which valves, like those schematically shown in FIG. 10,are connected by a tube or a conduit 8 to the inlet 102 of the device,preferably equipped with a three-way valve 9, and allow the lipoaspirateto be directly transferred through one or more steps, from the patientinto the device of the invention.

Such transfer may occur in a closed system, in which the biologicalmaterial never contacts the outside environment.

The device and method as described and claimed below use the suctionedlobular fat to obtain a cell suspension with a solid phase consisting ofcells, cell agglomerates having small and averagely constant sizes, anda liquid phase free from any impurity such as blood, oil, cell debrisand anesthetic liquid.

As shown in FIGS. 1 and 2, the device of the present invention iscomposed of a washing and separating container 1 having a washingchamber 101 for washing the liposuctioned material, which container 1has an inlet 102 and an outlet 103 for the liposuctioned material toenter the washing chamber 101 through the inlet 102 and for at leastpart of said material, particularly, in order of time, first the fluidcomponent and then the solid component, to exit said chamber 101 throughthe outlet 103, said washing chamber 101 including means formechanically forming an emulsion of fluid components, particularly oilobtained from broken adipocytes, blood and/or other sterile liquidsolutions.

Said emulsion-forming means consist of at least one stirring element104, such as balls or the like of equal or different sizes, forincreasing emulsion of liquid components when the washing and separatingcontainer 1 is subjected to stirring. In the device of the presentinvention, simple manual stirring of the device can afford separation ofthe liquid phase composed of the fluid emulsion from the solid phasecomposed of cells, cell fragments, cell aggregates.

Mechanical stirring may be also obviously provided, to simulate at leastthe manually exerted force.

These stirring elements 104 may be of relatively small size whencompared with the of the washing chamber 101, because if they were toolarge they could not move freely in the chamber 101 and might damage thecell material contained in that chamber 101. The weight of said stirringmembers should also be sufficient to form an emulsion of liquids withoutcausing cell wall breaking.

These stirring members 104 may be substantially spherical bodies, i.e.having continuous rounded exterior surfaces, and be hollow or solid, andmay rotate and move within the chamber 101 to assist gentle mixing ofthe liquid components.

The stirring members 104 are preferably made of a sterile material thatdoes not interact with the biological material contained in the washingchamber or with the solutions injected for washing the cell material:for example, they may be made of metal, which is easy to sterilize, evenat high temperatures, and prevents the stirring members from braking orbeing deformed upon collision with each other or with the inner walls ofthe washing chamber 101.

The rounded surfaces of the stirring members 104 may also facilitatesterilization, as they prevent the creation of debris build-up areas orbacterial proliferation areas on said members 104, which would hindercleaning and maintenance of sterile conditions. Preferably, the stirringmembers 104 consist of balls having a given weight and size, tofacilitate mutual emulsion of liquids without causing mechanicalbreaking of the cell walls during manual or mechanical stirring.

The emulsion of liquids is obtained due to the presence of the stirringmeans 104 which enhance the mixing action, while also gently stirringthe container to obtain emulsion of liquids, and preventing abruptmovements that might lead to cell breaking, such stirring not involvinga disintegration of the bonds between the liquid components.

Stirring members 104 of any shape and material, such as glass, may beobviously used.

The washing and separating container 1 preferably has a cylindricalshape, but it may be formed in any shape and size that might bothcontain a given amount of liposuctioned fat and ensure optimal handling.

In a preferred embodiment, the lipoaspirate in the washing chamber doesnot exceed ⅓ of the overall chamber capacity; the remaining volume isfilled with liquids.

For instance a container 1 having a 10 to 3000 cc volume may beprovided.

The preferred size is a container from 300 to 600 cc, with about 100 to250 cc lipoaspirate being treated therein.

As shown in the figures, the washing and separating container 1 iscomposed of a central tubular portion 111 with the washing chamber 101formed therein, and two closing terminals 112, 113 at the ends of saidtubular portion 111 such as caps 112, 113 or the like, the inlet 102 andoutlet 103 of the washing chamber 101 of the washing and separatingcontainer 1 consisting of a hole formed in each cap 112, 113,communicating with a connection terminal 1021, 1031 and/or closingvalves.

Obviously, said terminals 1021, 1031 and/or said at least one inlet 102and outlet 103 may be designed to be closed with sterile caps and/or beequipped with backflow preventing means, such as one-way valves, duringthe step of injecting and pressing fat into said container 1.

For instance, said inlet 102 and/or outlet 103 and/or said connectionterminals 1021, 1032 may be connected to three-way valves 9 like thoseschematically shown in FIG. 10.

The fat material is injected into the washing chamber 101 of thecontainer 1 through the opening 102 which is equipped, for instance,with a luer connector 1021 and/or a three-way valve 9.

Male, female and neutral luer connectors are known, and allowfluid-tight connection between two devices having said connectors. Luerconnectors are sold, for instance, by GVS, in their Web sitewww.gvs.com. Examples of syringes with a two-way valve are shown inwww.internationalpbi.it.

At least one filter 4 may be provided in the washing chamber 101 of thewashing and separating container 1 proximate to the outlet 103 whichallows the passage of the fluid component and/or the solid component ofthe fat and retains the stirring elements 104 in the washing chamber101.

In a variant embodiment of the present invention, said filter 4 mayconsist of a selectively permeable membrane that allows the passage ofat least part of the liquid phase, consisting of an oily component, ablood component, sterile solutions such as anesthetic liquid and/orsaline and retains the solid cell phase, consisting of cell fragments,whole cells and cell agglomerates, thereby allowing separation of theliquid phase of the emulsion from the solid phase.

Said selectively permeable membrane may consist of a net of fine meshes,which is smooth, i.e. with no projecting parts or irregular or sharpsurfaces that might damage cell walls, whose meshes or throughinterstices are smaller than the cell agglomerates contained in thewashing chamber 101 of the washing and separating container 1.

The meshes of the net that forms the selectively permeable membrane mayhave a size ranging from 1000 to 50 μm.

Such filter 4 is used to retain the stirring members 104 in the washingchamber 101 and prevent them from clogging the outlet 103 during washingand separation of the solid cell component from the emulsion.

In one preferred embodiment, as better described hereinafter, saidfilter 4 is replaced by a fine mesh cutting net that provides means forreducing the size of the solid component of the lipoaspirate that comesout of the washing chamber 101.

In one embodiment, at the opposite end, i.e. at the inlet 102, sizereducing means may be provided for reducing the size of the solidcomponent of liposuctioned fat 3, particularly cell macroagglomerates,to smaller cell agglomerates, i.e. having a size equal to or smallerthan a given value, which means consist of at least one series ofparallel or intersecting sheets or cutting wires made of sterilematerial, e.g. metal, to form at least one size reducing net, throughwhich the liposuctioned material is passed before entering the washingchamber 101 of the washing and separating container 1.

The container may be made of sterile plastic or glass, or anyway of atranslucent material preferably resistant to high temperatures andautoclave treatable, for injection therein of the liposuctioned fat.

As shown in FIGS. 4 and 5, said size reducing net for cell agglomerates3 is subtended in the washing chamber 102 proximate to the inlet 103 ofthe washing and separating container 1 in a position substantiallyperpendicular to the direction of the flow of fat entering the washingchamber 101 of said washing and separating container 1.

In a preferred embodiment of the present invention the sizes of thelipoaspirate are progressively reduced.

Such progressive reduction is obtained by forcing the lipoaspirate atleast once through the meshes or apertures of two or more size reducingnets at a given distance from each other.

In addition to the net proximate to the inlet 102, the washing andseparating container 1 has a second size reducing cutting net 6,subtended in the washing chamber 101 proximate to the outlet 103 of thewashing and separating container 1 in a position substantiallyperpendicular to the direction of the flow of fat exiting from thewashing chamber 101 of said washing and separating container 1, so thatthe lipoaspirate that enters the washing and separating chamber 101,before exiting from said chamber 101, may flow through two nets withprogressively finer meshes, the coarser meshes being provided in the netlocated proximate to the inlet 102.

Therefore, the two nets are located at a given distance from each other;particularly, they are located at the end sides of the washing andseparating container, separate from the washing and separating chamber101 that contains the stirring means 104 as well as a sufficient spaceto form an emulsion.

Considering the direction of the flow of lipoaspirate, the first net 3,i.e. the one located proximate to the inlet 102, stops the fibrouscomponents of the fat lobules and performs a first small reduction, e.g.provides cell agglomerates of a relatively homogeneous maximum size,i.e. having a diameter from 0.5 to 2 mm, whereas the second net 6,located proximate to the outlet 103, performs a further reduction of theagglomerates, that have been previously washed. For example, theagglomerates and/or the cells that come out on the washing andseparating container 1 may have a diameter of up to 10 um.

The second net 6, as a replacement of the filter 4, may be used to holdthe stirring members in the washing and separating chamber.

Such progressive reduction through the cutting meshes of multiple netsprovides a solid component to be injected which is composed ofagglomerates of very small diameter and, if needed, of individual cells.

The device of the present invention allows the lipoaspirate to bereduced to transplantable material of any size.

As the cell material passes through the second net 6 located proximateto the outlet 103, after the washing step performed in the washing andseparating chamber 103, said net having meshes or apertures of reducedsize, it may lead to oil formation, due to the break of adipocytes.

This oil may be removed by transferring the cell material into anothercontainer 1 having a washing and separating chamber 101, with or withoutsize reducing means.

As an alternative to or in combination with such transfer into a secondwashing and separating container, any residual oil/liquid may be removedby settling and/or centrifuging the treated material that comes out ofthe washing and separating chamber one or one and is placed in specialcontainers.

These containers may be one or more syringes held in a syringe holder.

As shown in FIGS. 12a and 12b , the nets located, with known techniquesand means, proximate to the inlet and outlet of the washing andseparating chamber 101, may have honeycomb cutting meshes.

In one embodiment, the area of each mesh of the net proximate to theinlet 102, is about 4 mm², whereas the area of each mesh of the netproximate to the outlet 103 is about 1 mm².

The reduction and homologation of the cell agglomerate size are providedby the sheets or the meshes of the net or preferably the two cuttingnets located in the container 1.

Each net has equal meshes or through interstices, having a diameter thatranges from 2000 μm to 50 μm, preferably from 1500 μm to 100 μm.

Since the meshes of the net 3 located proximate to the inlet 102 arelarger than the meshes of the second net 6 located proximate to theoutlet 103, a first size homologation and/or reduction of lipoaspirateagglomerates is obtained by passage through the meshes of the first net3, and a second increased reduction is obtained when the material iscaused to exit from the container 1, by being passed through the meshesof the second net 6.

In one embodiment, the interstices (meshes, apertures) of the sizereducing means for cell agglomerates, particularly in the net 6 locatedproximate to the outlet 103, have a diameter ranging from 750 to 50 μmand anyway sufficient to allow the passage of cell material, even ofindividual cells.

Obviously, lipoaspirate size reduction and/or washing may also not occurusing a single washing and separating container 1, having size reducingmeans therein, i.e. one, two or more nets, but using a succession of twoor more containers 1, adapted to be connected together. The meshes ofthe net/s 3, 6 of each container 1 may have different sizes from themeshes of the net/s located in another container 1 and/or the meshes ofthe net or nets of a container may have different sizes from each other.

Therefore, for progressive size reduction of lipoaspirate agglomerates,the material may be forced out of a device into a second device with awashing and separating chamber 1 and size reducing means having smallermeshes then the net or nets located in the previously used device.

As shown in FIGS. 6 and 7, at least one size reducing net for cellagglomerates 3 is subtended between an inlet 202 and an outlet 203 in areducing chamber 201 of a fat size reducing container 2, proximate tosaid inlet 202 of the size reducing container 2 for receiving the fatentering said reducing chamber 202, said container 2 also having anoutlet 203, for the liposuctioned fat to be injected through the inlet202, passed through the size reducing net 3 into the reducing chamber201 of the second container 2 and allowed to exit from said reducingchamber 201 through the outlet 203, said outlet 203 being designed to beconnected to the inlet 102 of the separation and collection container 1to provide communication between the size reducing chamber 201 of thesize reducing container 2 and the washing chamber 101 of the washing andseparating container 1.

Alternatively and preferably, the multiple step reduction may occur bycollecting the cell material into syringes, e.g. 10 cc syringes, andusing the latter to introduce the material into a second complete devicehaving progressively finer meshes.

As shown in FIGS. 6 and 7, the size reducing container 2 may be composedof two portions and half shells connected together, at least one of saidtwo portions, preferably the portion with the outlet 203, having aninner chamber 201 for collecting the size-reduced fat.

In one embodiment, said collecting container 2 may be designed to becomposed of two elements similar to the closing terminals of the washingchamber as described herein.

The two terminals may be connected to each other in a fixed or removablemanner, to form a size reducing container 2 with an inner size-reducingchamber 201.

Like the washing and separating chamber 1, the collecting container ispreferably of disposable type or may be removed for sterilization of theinternal elements and reuse.

Obviously, the size reducing chamber 201 maybe allowed to have varioussizes, to collect various amounts of size-reduced cell material, passingthrough at least the size reducing net 3.

Proximate to the outlet 203 a further cell agglomerate size reducing netmay be provided.

Particularly, two lipoaspirate size reducing nets may be provided in thesize reducing container 2, one proximate to the inlet 202 and the otherproximate to the outlet 203, said two nets having meshes of differentsizes: particularly, the net proximate to the inlet 202 has a largermeshes then the net proximate to the outlet 203. According to a variant,the device of the present invention is composed of at least one sizereducing container 2 and at least one washing and separating containerfor separating the fluid component from the solid component, said sizereducing container 2 having a size reducing chamber 201 with an inlet202 and an outlet 203 and a size reducing net 3 there between, theoutlet 203 having removable fluid-tight connection means 2021, 2031 forconnection with mating connection means 102 situated at the inlet 102 ofthe washing chamber 101 of the washing and separating container 1.

As shown in FIG. 7, the size reducing container 2 has an inlet 202 andan outlet 203 and a size reducing net 3 for the cell agglomerates thatform the solid component of fat, located in an intermediate positionbetween the inlet 202 and the outlet 203 or offset toward the inlet 203,and the downstream compartment, with reference to the direction ofinjection of fat, acts as a collection chamber for the size-reducedmaterial 202, said compartment having a predetermined volume.

The inlets 102, 202 and outlets 103, 203 of the size reducing container2 and/or the washing and separating container 1 have removablefluid-tight connection means, such as luer connectors or the like, forconnection to medical devices, such as syringes, bags or the like,having removable fluid-tight connection means compatible with the meanslocated at the openings 102, 202, 103, 203.

Obviously, the inlets 202 and outlets 203 of the size-reducing container2 may be designed to be closed with sterile caps and/or be equipped withbackflow preventing means, such as one-way valves, during sizereduction.

In order to allow the liposuctioned fat to flow into and out of the sizereducing chamber 201 of the size reducing container and/or the washingchamber 101 of the washing and separating container 1 and to allowremoval of the fluid component, i.e. the emulsion obtained by mechanicalstirring from the washing chamber 101, means are provided forcompressing or aspirating the fluid component and/or the solid componentof fat, such as syringes or the like, which means can be removably andfluid-tightly connected with the mating means 1021, 1031, 2021, 2031located at the inlets 102, 202 and the outlets 103, 203 of the sizereducing chamber 201 and/or the washing and separating chamber 101.

Obviously, the flow of fat material through the meshes of the sizereducing net 3, 4 may be facilitated by mixing said fat material withliquids, particularly a saline.

The size reducing net/s 3, 6 in the washing and separating container 1and/or the net/s 3 in the size reducing container 2 may be each designedto have interstices or meshes of different sizes, so that the nets in acontainer have meshes of different sizes from each other and/or from themeshes of the net/s in another container, so that the cell agglomeratesize reduction aimed at obtaining cell agglomerates equal to or smallerthan a given value, occurs in multiple steps through the nets in saidcontainers 1 and/or 2.

Therefore, the purpose of this part of the device of the invention is toreduce the size of adipose tissue lobules by forcing them through aspecial cutting net or parallel cutting sheets, preferably through twonets, and form a cell suspension with cells and/or cell agglomerates,particularly adipocytes, having reduced and averagely uniform sizes, oranyway agglomerates having equal or smaller sizes than a predeterminedvalue, said cell suspension being adapted for use in a latertransplantation step using particularly thin needles or cannulas, whileavoiding clogging thereof.

The purpose of the size reducing net/s is also to increase the amount ofperipheral stem cells that may contact the tissue to be treated afterinjection, which stem cells adhere to the outer surfaces of the cellagglomerates obtained by reducing the lipoaspirate mass.

With the above device, the sizes of cell agglomerates, upon passagethrough the size reducing means, are substantially identical and rangefrom 2000 μm to 50 μm, preferably from 1500 μm to 100 μm.

As shown in the figures, the washing and separating container 1 iscomposed of a central tubular body 111 and two closing terminals 112,113 or caps, which are or can be fixed to the ends of the central body111.

Said central portion 111 is shown as having a cylindrical shape, but maybe of any shape and size.

The washing container 101 has such a size as to allow handling thereofby one hand.

In one variant, as shown in FIGS. 2, 3, 4 and 5, a widened cup-shapedsection 115 is provided at one end of the central tubular portion 111,which section is a widened axial end of said tubular portion, whoseinside diameter is larger than that of the central portion 111.

One closing terminal 112 has an opening adapted to engage with the endof the central portion, whereas a second closing terminal 113 has anaxial cylindrical extension 116 for engagement in the cup-shaped seat115 of the central portion 111, by abutment against an. outer annularradial abutment shoulder 117.

At the contact surfaces of the axial extension 116 of the closingterminal 113 and the widened cup-shaped portion 115 at one end of thewere portion 111, and between the exterior surface of the end side ofthe central portion and the interior surfaces of the opening of asecond. closing terminal 112 in contact therewith, means for fluid-tightconnection, such as o-rings or the like may be provided, which form, incombination with the above described components, i.e. the closingterminals 112, 113 and with the central portion 111, a completelysterile washing chamber 101, isolated from the outside environment.Indents and grooves may be further provided on these contact surfaces,for ensuring fixation of these terminals 112, 113 on the tubular body111.

Said closing terminals 112, 113 may be designed to be removably fixed tothe central tubular body 111, but said washing and separating container1 may be also designed to be removable to allow separation of one orboth terminals from the tubular portion 111. Therefore, this willprovide a disposable washing and separating container 1, which isadapted to be sterilized also in its inner washing chamber 101, andhence to be reusable.

As shown in FIGS. 13a, 13b, 13c , the closing terminals 112, 113 and thecentral tubular portion may have very simple constructions: the twoclosing terminals may be in the form of caps fixedly or removablymounted to the ends of the tubular body 111.

These terminals may have shoulders on the side facing toward the washingchamber, to prevent deformation of the nets, that may be thin anddelicate.

These closing terminals 112, 113 have apertures, such of through holes,formed at the longitudinal center axis of the tubular portion, whichapertures have closing valves and/or fluid-tight connection means 1021,1031 such as luer connectors, or snap-fit or screw connectors or thelike, or multiple-way valves, providing connection of the washingchamber 101 with one or more medical devices, possibly at the same time,such as syringes, bags or the like, or with the side reducing chamber201 of the size reducing container, through respective inlets 202and/or: outlets 203 having mating connection means 2021, 2031.

As shown in the figures, a filter 4 is provided at the outlet 103, atthe connection between the closing terminal 113 and the end side of thecentral tubular portion, inside the washing chamber, substantiallyperpendicular to the longitudinal axis of the central tubular portion111, which filter maintains the stirring members 104 within the washingchamber 101, to prevent them from clogging any opening, particularly theoutlet 133, when the washing and separating container 2 is verticallyoriented relative to the ground for removal of the liquid emulsion. Asmentioned above, the washing and separating container 1 may be alsodesigned to be provided, at the inlet 102, with size reducing means 3for reducing the size of lobular fat yielded from liposuction, intocells and cell agglomerates, particularly adipocytes and stem cells, ofsmaller and identical or similar sizes.

As mentioned above, instead of or in addition to the filter 4, a sizereduction means for the liposuctioned material may be provided, such asa lipoaspirate size reducing net, which lipoaspirate is further dividedinto smaller agglomerates before coming out of the washing andseparating chamber 101.

Therefore, at the connection between the closing terminal 112 and theend side of the central tubular portion, a size reducing net is providedin the washing chamber proximate to the inlet 102, substantiallyperpendicular to the longitudinal axis of the central tubular portion111, which net reduces the size of a macroagglomerates to apredetermined value, which macroagglomerates are injected or pushed intothe washing and separating chamber through the inlet 102.

A further size reducing net may be provided at the connection betweenthe closing terminal 113 and the end side of the central tubularportion, in the washing chamber 101 proximate to the outlet 103,substantially perpendicular to the longitudinal axis of the centraltubular portion 111.

In this preferred embodiment, the passage through their first sizereducing net, proximate to the inlet 102, fragments the pieces ofconnective tissue of the lipoaspirate and/or provides a first coarsereduction of the lipoaspirate agglomerate size, whereas the passagethrough the second size reducing net located proximate to the outlet 103provides cell agglomerates whose size is equal to or smaller than agiven value, said nets having meshes of different sizes, particularlythe first net, with reference to the direction of fat flow in thewashing and separating container 1, having larger meshes than the secondnet.

The adipose tissue mass prepared by the above described device, i.e. theadipose tissue that underwent size reduction through the size reducingnet/s and/or washing and separation of the solid component from theliquid component, is mainly composed of adipocytes, but also other typesof perfectly healthy and viable cells that may be found in alipoaspirate, and may be used for transplantation in face and/or bodyremodeling procedures.

The present invention addresses a method of treating ‘or’ preventinginjuries or diseases in a patient, particularly a method of treatingvolume deficiencies in the body and face, improving skin trophism and/orof biological stimulation, which method includes:

-   -   at least one step of extracting biological material from donor        areas of the patient, particularly adipose tissue extracted by        liposuction,    -   at least one step of treating said material,    -   at least one step of injecting the treated material into a        patient.

Before the injection step, a step of collection and storage of thebiological material may be obviously provided.

The treatment step includes at least one size reduction step forreducing the size of the extracted material and/or at least one washingand separation step for washing and separating the liquid phase from thesolid cell phase, said treatment step being carried out using the deviceand method of the present invention.

In the present method, the donor patient is also the receiver patient inwhich the injection step is carried out.

Nevertheless, the donor and a receiver may also be different persons.

Obviously, the device and method for treating the tissue and the methodof treating the patient may involve the use of a tissue other thanadipose tissue.

An exemplary procedure for transplantation of the biological materialprepared by the above device will be now described.

The material prepared using the above described device may be stored inone or more sterile containers, e.g. syringes, and allowed to settle andpossibly centrifuged to separate the solid component from any residualoil or solutions.

The cell material prepared using the device and method of the presentinvention may be injected into any type of tissue and with any suitableprocedure.

Once the receiving areas have been designed and accurately disinfected,the needle or microcannula of the syringe containing the prepared cellsuspension is introduced into the subcutaneous or muscular tissue,thereby creating at three-dimensional net of tunnels for injection ofvery small amounts of cell agglomerates.

This step is preferably carried out using a disposable sterileblunted-end cannula with a luer connector, having a very small diameter.

The small size of the tunnels formed in the treated tissues facilitatesintegration of the cells, and cell agglomerates in the interstitialspaces of the subcutaneous tissue or in the muscular tissue, therebyreducing surgical trauma and facilitating quick return to normalconsistency of tissues undergoing volume increasing and remodelingtreatments.

Therefore, the formation of vary small-diameter tunnels in the tissuesoptimizes tissue volume reconstruction and/or biological stimulationresults.

The reduction of fat lobule sizes by dividing such lobules into cellagglomerates affords a lager contact surface between the injected massand the tissues being treated, thereby facilitating biologicalstimulation of the relevant areas and integration of the transplantedadipose tissue.

The use of these particularly thin cannulas, possibly of less than 1 mm,is allowed by the reduction of the size of the liposuctioned lobularfat, which occurs in at least one size reducing container 2 and/or atleast one washing and separating container 1 having size reducing meansconsisting of at least one series of parallel sheets and/or at least onenet of wires or thin sheets, preferably at least two nets, at least oneof said two nets having meshes of very small sizes, which allow thepassage of cell agglomerates or even individual cells having sizes ofthe micron order.

Should no fat size reduction occur, the cannulas that are used intransplantation procedures would become clogged.

As an alternative to sterile cannulas with a blunted end, helical orspiral-shaped cannulas 5 having a pointed or blunted end like the one asshown in FIG. 12 may be used for transplantation of adipose cells.

A helical cannula 5 is particularly useful for injection into organs inwhich a few penetration steps of the transplantation needle arerequired: the corkscrew needle affords maximized cell material densitydeposited along a single path.

Such type of cannula allows transplantation of adipose tissue cellagglomerates even in high-consistency tissues or particularly delicatetissues, such as scar tissue, bone, cartilage, myocardium or in otherorgans, through a single injection point that allows treatment of acertain volume of tissue.

The cannula is introduced with a rotary motion to allow the helix toenter the tissue. Then, the cannula is extracted in the same mannerwhile injecting cell agglomerates or clusters of adipose tissues. Thiswill considerably increase the amount of transplanted adipose tissue perunit volume, as compared with individual injection by a rectilinearneedle, and hence the volume of treated volume without reducing thecontact surface between the injected adipose tissue and the receivingtissue thereby increasing the vascularization potential of injectedcells, as the adipose tissue is released as strips or very thin beads ofcells, due to the small diameter of the cannula, and in a spiral path.

The use of these cannulas, that allow the prepared adipose tissue,containing, in addition to adipocytes, other types of cells includingstem cells, to be released in a spiral path into the tissues to betreated, is particularly suitable when there is no way to performColeman filling, i.e. to form a net of tunnels in the tissue to betreated due, for instance, to excessive consistency of the tissue to betreated, or when tissue trauma is undesired, such as in myocardiumtreatment. The present invention also relates to a tissue fortransplantation, e.g. for autotransplantation or heterotransplantation,which tissue is composed of cells, possibly cell fragments and/or cellagglomerates, and is obtained using the above described device and/ormethod.

The tissue is composed of cell material of uniform size, with averagediameters ranging from 10 μm to 2 mm.

Preferably 0.05 to 1.5 mm, especially about 0.75-0.5 mm or less (up to10 μm).

If needed, the present device may also provide tissue composed ofindividual cells.

In a preferred embodiment, the tissue is an adipose tissue mainlycomposed of adipocytes and stem cells.

Said stem cells are adapted to be used to create any cell type, such aschondrocytes, osteocytes, adipocytes, nerve cells.

After preparation, this adipose tissue has a small liquid fraction, freeof any impurity, which is mixed with cell agglomerates and is sufficientto facilitate introduction of the cells into the tissues to be treated.

Said liquid fraction may be about 50% by weight of the adipose tissueprepared for transplantation.

The present invention also obviously relates to a tissue having one ormore of the above characteristics, but other than adipose tissue.

The present invention also relates to a preferably sterile anddisposable kit, adapted for use both in outpatient and surgicalsettings.

The device or kit of the present invention has two- or three-wayconnectors, with or without valves, connection tubes and syringes, andvarious containers (e.g. a bag for the washing saline and a bag forcollecting washing waste).

The kit may be used to treating cell material of any type, preferablyfat for auto- or hetero-transplantation 1 and comprises at least onecontainer 1 for washing the solid component of fat and separating saidsolid component from the fluid component, said washing and separatingcontainer 1 being formed as described above.

This kit, which is composed of at least one washing and separatingcontainer having size reducing means in the washing chamber 101 isparticularly advantageous when used in the field of cosmetic treatment,e.g. in outpatient settings, as it provides a simple perfectly steriledevice, to be used for quickly treating the material extracted from thepatient, with no risk of cell material contamination due to contact withthe outside environment.

The kit allows treatment of biological material, from the first tissuesuctioning step to the last injection step, in a completely closedsystem, which allows the material to be treated without causing it toever contact the outside environment and/or without using means thatmight contaminate it.

This is allowed through the use of one or more tubes, syringes, bags,multiple-way valves, luer connectors and obviously one or more washingand separating containers 1 (and possibly one or more size reducingcontainers 2), which are sterile and connected or connectable togetherin a fluid-tight manner.

Therefore, the system that provides withdrawal of biological material,treatment of the material, storage, injection of the biological materialis a system that is completely isolated from the outside environment inall of its steps, from withdrawal from the patient to injection into thereceiving patient.

One or more of the system components may be mechanized by connection tospecial equipment, so that one or more steps of the process thatincludes suctioning and/or treatment of the biological material and/orinjection may be carried out without requiring any action by anoperator.

This kit is particularly suitable for minor aesthetic surgeryprocedures. In one preferred embodiment, the kit comprises one or morecontainers 1 having at least one size reducing net, preferably two sizereducing nets, as described above. Each container 1 of the kit may bedesigned to have nets with meshes of different sizes from those of theother containers 1 so that, by forcing the material through the meshesof the nets of multiple containers, cell agglomerates are progressivelysize-reduced to the desired value allowing transplantation.

Such progressive reduction avoids the risk of clogging the meshes of thenets and prevents the treatment process from being slowed down.

As an alternative, a kit may be provided which comprises:

-   -   at least one container 1 for washing the solid component of fat        and separating said solid component from the fluid component,        possibly having at least one size reducing net or sheets 3,    -   at least one size reducing container 2 formed as described above        and adapted to be connected to said container 1.

In one embodiment, the kit has two or more size reducing containers 2,which are adapted to be alternately connected by their outlets 203 orinlets 202 to the inlet 102 or outlet 103 of the washing and separatingcontainer 1, a predetermined amount of fat of reduced size being storedand preserved in sterile conditions in the reducing chamber 201 of eachsize reducing container 2.

A kit having a set of size reducing containers 2 may be also provided,which is composed of at least two size reducing containers 2 havingdifferent sizes in terms of the meshes of the size reducing net 3 and/orthe volume of fat of reduced size contained in the reducing chamber 201.

For example, the kit may be designed to contain two or more sizereducing containers 2 having size reducing means 3 through which thecell material may be forced, each having a net 3 with meshes ofdifferent sizes.

The provision of two or more size reducing containers 2 allows treatmentof a large amount of fat with no risk of clogging the meshes of the sizereducing net and hence of slowing down the material treatment process.

The washing and separating containers 1 and/or the size reducingcontainers 2 may be of disposable type or may be formed in such a manneras to allow complete sterilisation and later reuse thereof.

The kit of the present invention may also include, alternatively or incombination:

-   -   one or more disposable sterile syringes with different volumes,    -   one or more sterile pointed needles or sterile lanceolate blades        of different particular sizes to allow transcutaneous        introduction of cannulae for anaesthesia, removal and        transplantation,    -   one or more disposable sterile cannulae having a pointed or        blunted end, at least one of which has a very small diameter, of        the order of 1 mm,    -   one or more, one- or multiple-way valves, with or without check        valves, e.g. three-way valves to be connected to the inlets 102,        202 and/or outlets 103, 203 of the containers 1 and/or 2,    -   one or more sterile tubes with luer connectors, allowing the        passage of biological material from one component of the kit to        another (e.g. from the syringe to the washing and separating        chamber 1 or from a saline bag to the washing and separating        chamber)    -   means or containers, such as syringes, to allow settling and/or        possibly centrifugation of the fat material prepared using the        above described the device, which fat material may be        distributed into one or more sterile containers, to provide        additional separation of the solid component, which floats after        settling on the residual liquid/oily component to be removed        before transplantation,    -   means for preserving the biological material so prepared and        ready for transplantation, e.g. means for cryopreservation        thereof in a closed environment simulating a clean room, i.e. a        container having controlled conditions, e.g. in terms of        particulate pollution, pressure and temperature.

As schematically shown in FIG. 10, the kit may comprise one or moredevices such as one or more washing and separating and size reducingcontainers 1, syringes 8 for suctioning material from the patient,syringes 12 for collecting/injecting the treated the biologicalmaterial, washing liquid bags 10 (e.g. a bag containing saline), bags 11for collecting waste material, which are or can be connected together bysterile tubes, multiple-way valves, luer connectors.

Instead of or in addition to said small diameter cannulas, the kit mayinclude at least one helical or spiral-shaped cannula 5 with a pointedor blunted end allowing, as described above, transplantation of the cellmass of adipose tissue into high-consistency or particularly delicatetissues by increasing the volume of treated tissue with a singleinjection point.

The kit may also include an instrument for locking syringes duringsuction to temporarily prevent the plunger from bouncing back, e.g.during liposuction, and allow less traumatic adipose tissue suction.

The kit may further comprise a spring-biased mechanism for imparting areciprocating motion to the syringe piston, which mechanism is connectedto the two-way valve and affords quick withdrawal of adipose tissue,which adipose tissue is conveyed to the washing and separating chamber101 without contacting the outside environment, through a tube havingluer connectors at its ends.

The provision of a three-way valve at the inlet 102 of the chamberfurther allows injection of a washing solution into said chamber withoutdisconnecting the withdrawing syringe.

The syringes in the kit may be made of plastic, preferably with a luerconnector, or the like, and have various volumes.

The following may be used, by way of example:

-   -   10 to 60 cc syringes for local anesthesia injection,    -   5 cc syringes with needle for creating a wheal of anesthetic,    -   10 cc or larger volume syringes, connected to sterile 1.5 to 3        mm diameter cannulae for drawing adipose tissue from donor        areas,    -   1 to 5 cc syringes for tissue transplantation.

Therefore, the treating method that may be carried out with the deviceof the present invention allows preparation of an adipose extract, e.g.obtained by liposuction, which is in the form of a mixture of fluidmaterials and cell fragments and one or more cell macroagglomerates ofheterogeneous sizes, in a cell suspension containing cell agglomerates,particularly adipocyte agglomerates, with smaller and identical orsimilar sizes, in any case, smaller than a given value, to allowtransplantation into areas of the face or body of the patient, requiringa filling procedure with minor trauma, and accompanied by biologicalstimulation of the tissues involved in the procedure.

The method may be carried out in a closed system to avoid contaminationof the cell material before administration thereof to a patient.

Obviously, the adipose tissue to be transplanted may be obtained notonly by liposuction but also using other known techniques.

As described above, agglomerates may be obtained with an average size of500 μm, or smaller, i.e. about 100-10 μm, depending on the sizes of themeshes or apertures of the size reducing means that was used forliposuctioned material size reduction.

Therefore, the preparation of fat material involves the division of saidfat material into cell fragments, cells or cell agglomerates that aresmaller than the suctioned macroagglomerates.

Such division, as mentioned above, enhances the activity of stem cells,as it creates a favorable microenvironment facilitating contact of thestem cells with the tissue in which transplantation occurs.

In the present invention, for the cell agglomerates to be divided tosizes equal to or smaller than a given value, progressive reduction oflipoaspirate size is preferred, which means that fat is forced at leastonce through at least one cutting net of intersecting or—parallel wiresor sheets, preferably through two nets located at a given distance fromeach other, said nets having meshes of different sizes.

Before and/or after size reduction, the fat may be obviously washed onceor multiple times with a sterile washing solution.

The cell agglomerates so obtained undergo an additional treatment, whichinvolves washing with sterile solutions and separation of the cellcomponent from the liquid phases, i.e. blood, oil that comes out of thebreak of adipocytes, any anesthetic solutions in use, and the solutionin which said agglomerates have been mixed, e.g. a saline.

Washing and separation are allowed by the use of a container 1 with awashing chamber containing stirring members 104 such as balls or thelike which, by stirring the container 1, can form an emulsion of theliquid components contained in said washing chamber.

Washing of cell agglomerates in one or more washing and separatingcontainers may continue until the liquid waste phase that comes out ofthe outlet 103 is perfectly clear.

Advantageously, since progressive size reduction of the lipoaspirate isobtained by forcing it through at least two nets located at a givendistance from each other in a washing and separating container, i.e. atthe ends of the central tubular portion, cell agglomerates are washed afirst time after a first step of separation of adipose tissue lobules ormacroagglomerates and reduction/homologation of the sizes of saidagglomerates below a predetermined value, since the passage through theadipose tissue size reducing means 3, which is obtained by applyingpressure on said adipose tissue, may cause the cell walls of adipocytesto break, with the formation of oil that has to be removed from the cellsuspension containing cell agglomerates, to ensure successfultransplantation of said tissue.

One or more later washing steps will be performed after a second orlater size reducing steps, for obtaining a solid component of adequatesize for transplantation.

Washing and separation of the cell component from the liquid phase ofthe extracted fat in the above described container 1, may also occurbefore the cell aggregate size reduction steps.

The method of the present invention includes, instead of or in additionto the dividing step, at least one step of washing the cell aggregates,which is carried out at the same time as a step of separating the fluidcomponent, in emulsion form, from the solid component.

FIGS. 8a-8d and 10 schematically illustrate the method.

In a first step, area is exhausted from the washing and separatingcontainer 1, and the whole inner volume is filled with a liquid.

For example, the air to be exhausted before use of the device may beremoved by aspirating saline with the syringe 7, by causing it to enterthe bag 10 by gravity and by causing air to exit from the syringe 12(after removing the syringe cap) and/or from the container 11 (which maybe equipped with an openable exhaust valve).

The washing and separating container 1 is vertically oriented with theoutlet 103 open and facing upwards: liquid is introduced into thecontainer 1 and air is exhausted therefrom through the inlet 102.

The liquid may be a saline contained in a bag 10 connected via a tube tothe opening of the chamber, which has a three-way valve.

Then, the lipoaspirate is injected into the washing and separatingchamber 101.

The lipoaspirate may be injected directly from the suction syringe ofthe container 1 or through a completely closed system as shown in FIG.10.

Syringes of any volume may be used 10 cc syringes are preferred.

The fat material is injected into the chamber 101 through a suctionsyringe used for withdrawal, e.g. a two-way syringe equipped with avalve, directly or through a tube connected to the syringe and to theopening of the container and the fat material is pushed into saidchamber by the pressure action exerted by the piston of the syringe.

In this step, the container is preferably held in a vertical positionwith the outlet 103 facing downwards, to the ground.

The presence of size reducing means proximate to the inlet 102 providesa first lipoaspirate size reduction/homologation.

A hydraulic force may be applied to the fat material by a washing fluidunder pressure, which is injected into the washing and separatingchamber 101 and forces the fat material into and out of the washingchamber 101.

The adipose agglomerate injected into the separating and washingcontainer 1 through the inlet 102, may be repeatedly washed by pressureinjection of liquid materials such as sterile salines into said innerwashing chamber 101 to obtain a high-purity cell agglomerate, free ofany oil, blood and of any solution used during withdrawal.

The washing and separating step in which said fluid materials areseparated from said agglomerates occurs by:

-   -   at least one injection of a sterile washing solution, e.g.        saline, into the washing chamber 101 containing the fat        material, of a washing and separating container 1, which washing        chamber 101 contains at least one stirring element 104. In this        step, the container is held in a vertical position with the        outlet 103 facing toward and parallel to the ground    -   manual or mechanical stirring, manual stirring being sufficient,        of said washing and separating container 1 to facilitate        emulsion of fluid components, particularly the oily component        and blood with the sterile fluid substances; the emulsion being        formed by orienting the washing and separating container in a        horizontal position, i.e. approximately parallel to the ground        (Figure Bb),    -   arrangement of the washing and separating container 1 in a        vertical position relative to the floor, with the outlet 103        facing downwards, to obtain a stratification of the solid        components on the liquid emulsion which constitute the fat        contained in the washing chamber 101, particularly to obtain a        solid component composed of cell fragments, cells and one or        more cell agglomerates floating on an emulsion of the fluid        components in the lower portion of the washing chamber 101 in        contact with the outlet 103 of the washing and separating        container 1,    -   discharge of the emulsion of fluid components (i.e.        oils/liquids) from the washing chamber 101 through the outlet        103 of the washing and separating container 1 (FIG. 8c ). The        emulsion is forced out by injection of washing fluid through the        opening 102, with a given pressure.

Since the container has a cylindrical shape, its horizontal positionmeans that its longer axis passes through the end sides parallel to theground, whereas its vertical position means that the cylinder isoriented with its longer axis perpendicular to the ground.

The emulsion is collected in another container 11 which is fluid-tightlyconnected by suitable means to said opening 103, to preventcontamination of both the outside environment and the cell materialcontained in the chamber 101.

Therefore in the washing step, the washing and separating container 1containing fat mixed with a sterile solution, e.g. either salineinjected with a syringe through the inlet 102 or saline withdrawn from abag, is stirred with a force that does not cause the cell walls to breakbut is sufficient to form an emulsion, i.e. a dispersion of tiny oildrops within the washing fluid, due to the presence of the balls 104.

The container is stirred to form the emulsion with the container 1horizontally oriented.

Obviously in this step the openings, i.e. at least one inlet 102 and/orone outlet 103 of the container 1 are closed to prevent any leakage ofmaterial.

At the end of the container stirring step, the container is moved to avertical position and the different densities of the materials that formthe lipoaspirate create one solid layer of cells and cell fragments inthe washing chamber 101, which layer floats on the emulsion of liquids.

The washing step is repeated by injecting sterile washing solutions intothe chamber, with subsequent emulsion formation (by stirring thecontainer with the inlet and the outlet closed) and discharge of theemulsion (by the injection of a clean washing solution), until theoutflowing liquid-oil-blood emulsion appears to be free of any impuritysuch as blood and oil.

Therefore, the discharge of fluid component emulsions may be repeated.

Such discharge of the emulsion is obtained by the flow of aphysiological liquid caused by gravity, which flow allows removal ofliquid components (oil/liquid) through a density gradient, at least fora given time interval, i.e. until there is emulsion between tiny oildrops and liquid, which is sufficient each time, i.e. for each washingcycle, to remove a considerable amount of waste liquids (particularlyoil).

The water/oil emulsion is eliminated from the container through adensity gradient, following the outflowing liquids (the flow from theinlet 102 to the outlet 103) obviously provided that said washing flowis sufficient.

As shown in FIG. 9, the oil/liquid emulsion is discharged.

Oil can be discharged as long as it is part of an emulsion.

What is discharged is an emulsion of tiny oil drops and washing liquid.The cell mass floats on said emulsion.

Therefore, the emulsion is required to allow elimination of impurities,such as oil, in the lipoaspirate.

At the end of the washing step, the solid component floats on the cleanwashing solution.

Therefore, at the end of the washing and separating step, the materialin the chamber 101 of the container may be used for transplantation.

Adipose tissue washing is an important step, as it allows removal of oilresulting from the break of the cell walls of adipocytes duringmechanical withdrawal of adipose tissue, using cannulas or needles, fromdonor areas and during passage of fat, under pressure, through the sizereducing net 3.

The solid component does not remain in the device 1 but is recovered,preferably after further size reduction performed at the outlet 103, bya hydraulic thrust exerted from the inlet 102.

The solid component is discharged from the washing and separatingcontainer by vertically orienting said container with the outlet 103,with a size reducing net 6 preferably provided proximate thereto, facingupwards, so that the solid component floats on the washing solution(FIG. 8d ).

Therefore, the solid component to be discharged is located next to theoutlet 103.

The solid component is pushed out through the second net 6, by injectionof the washing solution through the inlet 102.

It was found that what is discharged is first an aqueous solution withlittle cell material, then a solution rich with cell material andfinally a solution with little cell material.

One or more containers may be connected to the outlet 103, one after theother, for instance syringes 12, for collecting the prepared biologicalmaterial mixed with liquid.

Such material has undergone further reduction/homologation through thenet 6.

The container 12 are allowed to settle to obtain the separation of thesolid component from the residual liquid component.

Instead of or in addition to the above, separation may occur bycentrifugation.

The biological material so prepared is preserved or preferablycryopreserved in a closed environment, simulating a clean room.

Obviously, the material stored in the container 12 may be designed to betreated again in a washing and separating container 1 and/or a sizereducing container 2, before being finally preserved and/or reinjected.

The whole material treating system is closed, from withdrawal from thepatient to reinjection. This characteristic is particularly importantfor banking of biological tissues, i.e. preservation thereof inbiological banks.

Therefore, the biological material conveyed into the container 12 hasalways been in a sterilized close system, never in contact with air.

Thus, said material may be directly cryopreserved with no further changerequesting the use of a clean chamber.

As described above, multiple mutually connectable washing and separatingcontainers may be provided, for progressive reduction and/or washing ofthe lipoaspirate.

The device of the present invention allows simple, quick and inexpensivetreatment of cell extracts in the form of cell macroagglomerates mixedwith a liquid phase, which provide cell aggregates of substantiallyidentical whole and viable cells of predetermined size, anyway smallerthan a predetermined value, which agglomerates are separate from theliquid component, in the form of emulsion, containing waste liquids.These agglomerates may be used for transplantation.

Obviously, the material yielded from treatment of adipose lobules maycontain not only cell aggregates but also individual cells and cellfragments to be used as a biological filler.

Furthermore, the liposuctioned material contains not only adipocytes butalso other types of cells, such of stem cells.

The treating method implemented by the device does not involve the useof enzymes or other components that can have a chemical action on theliposuctioned material, or a biological action on agglomerate composingcells, but uses the possibility of changing the size of cellagglomerates, to obtain a larger exposed cell surface, that may contactthe tissues treated during transplantation. Particularly, solid materialfor injection is provided, which also forms an optimal in vivomicroenvironment for the action of stem cells in the areas in which saidtreated material is reinjected, which stem cells are contained in thesuctioned material.

Therefore, the method of the present invention simply and inexpensivelyprovides biologically active injectable biological material from thematerial extracted from the patient, also due to the presence of stemcells, and requires neither the use of chemicals such as emulsifiers orenzymes, nor in vitro culture steps. Furthermore, these very small cellagglomerates may be transplanted using very thin cannulas, which reducesurgical trauma and optimizes tissue integration.

Also, the use of a device of the present invention provides a closedsystem that isolates the biological material from the outsideenvironment and allows it to be prepared for use in a very short time,thereby reducing possible contamination risks.

The device, kit and method as disclosed above, which form the subject ofthe present invention, may be obviously used not only for preparingadipose tissue to be transplanted, but also for preparing any type ofcell agglomerate that is required to have a high purity level for use.

I claim:
 1. A method of preparing adipose tissue for transplantationfrom lobular fat material extracted from a body by liposuction, said fatmaterial comprising (a) a fluid component comprising one or more of anoily component, a blood component and sterile solutions, and (b) a solidcomponent comprising tissue fragments and one or more cellmacroagglomerates of heterogeneous size, said method comprising washingthe solid component at least once, and separating the fluid component,in emulsion form, from the solid component, wherein said washing andseparating steps are carried out at the same time, and wherein saidwashing and separating steps include: injecting at least one sterilewashing solution into a washing chamber containing the fat material, ofa washing and separating container, to wash the fat material, whereinthe washing chamber contains an outlet and a plurality of stirringelements each having a relatively small size when compared with a sizeof the washing chamber; manually or mechanically stirring the pluralityof stirring elements in said washing and separating container tofacilitate the emulsion of the fluid components and the at least onesterile washing solution, and to separate the liquid emulsion from thesolid component, wherein the liquid emulsion remains separate from thesolid component without using enzymes or chemicals; arranging thewashing and separating container in a vertical position relative to theground, with the outlet facing downwards, to obtain a stratification ofthe solid components on the liquid emulsion, which constitutes the fatmaterial contained in the washing chamber, which results in said solidcomponent floating on the liquid emulsion of the fluid components thatare present in a lower portion of the washing chamber and in contactwith said outlet of the washing and separating container; anddischarging of the emulsion of fluid components from the washing chamberthrough the outlet of the washing and separating container, andfacilitating the location of the solid component next to the outlet anddischarging the solid component from the outlet, wherein said method iscarried out without using chemicals, enzymes, or in vitro culture. 2.The method according to claim 1, wherein, during injection of saidsterile washing solution, the washing and separating container is heldin a vertical position with said outlet facing toward the ground.
 3. Themethod according to claim 1, further comprising a step of dividing saidfat material into cell agglomerates smaller than said macroagglomerates,so that said cell agglomerates are as large as or smaller than apredetermined size, and so that they are on average of the same size,wherein this size reducing step is carried out by causing said extractedlobular fat to pass at least once through size reducing means located insaid washing and separating container or in a size-reducing container.4. The method according to claim 3, wherein the size reducing meanscomprises at least one series of parallel or intersecting sheets orwires that form at least one size reducing net.
 5. The method accordingto claim 1, further comprising a separating phase carried out after saidmanual or mechanical stirring step by means of at least one filter. 6.The method according to claim 5, wherein said filter is provided in thewashing chamber of the washing and separating container proximate to theoutlet, for allowing the passage of the fluid component and/or the solidcomponent of the fat material and retaining the stirring element in thewashing chamber.
 7. The method according to claim 1, wherein theplurality of stirring elements comprises at least a spherical body orball freely movable within the washing chamber.
 8. The method accordingto claim 1, wherein the plurality of stirring elements comprises aplurality of spherical bodies or balls movable within the washingchamber.
 9. The method according to claim 1, further comprising thesteps of: withdrawing adipose tissue from a body of a patient byliposuction performed with a sterile syringe connected to a cannula; andtransferring the liposuctioned material from the patient into saidwashing chamber.
 10. The method according to claim 1, further comprisingthe step of collecting the emulsion of fluid components discharged fromthe washing and separating container in a container which isfluid-tightly connected by suitable means to said outlet of the washingand separating container or of a size reducing container, therebypreventing contamination of both the outside environment and the cellmaterial contained in the chamber.
 11. The method according to claim 10,wherein the discharge of the emulsion in a further container is obtainedby the flow of a physiological liquid caused by gravity.
 12. The methodaccording to claim 1, wherein the plurality of stirring elements freelymoves in the washing chamber without damaging the cell materialcontained in the washing chamber.